Analysis of a catalog of S-AS pairs in the human and mouse genomes revealed several putative roles for natural antisense transcripts and showed that some are artifacts of cDNA library construction. A significant number of genes in mammalian genomes are being found to have natural antisense transcripts (NATs). These sense-antisense (S-AS) pairs are believed to be involved in several cellular phenomena.Here, we generated a catalog of S-AS pairs occurring in the human and mouse genomes by analyzing different sources of expressed sequences available in the public domain plus 122 massively parallel signature sequencing (MPSS) libraries from a variety of human and mouse tissues. Using this dataset of almost 20,000 S-AS pairs in both genomes we investigated, in a computational and experimental way, several putative roles that have been assigned to NATs, including gene expression regulation. Furthermore, these global analyses allowed us to better dissect and propose new roles for NATs. Surprisingly, we found that a significant fraction of NATs are artifacts produced by genomic priming during cDNA library construction.We propose an evolutionary and functional model in which alternative polyadenylation and retroposition account for the origin of a significant number of functional S-AS pairs in mammalian genomes. Cis-NATs are usually related in a one-to-one fashion to the sense transcript, whereas a single trans-NAT may target several sense transcripts [Natural antisense RNAs ) are endogenous transcripts with sequence complementarity to other transcripts. There are two types of NATs in eukaryotic genomes: nscripts -3. In thWhen evaluated globally, several features related to the distribution of NATs strongly suggest they have a prominent role in antisense regulation in gene expression -7. For iNATs were initially identified in studies looking at individual genes. However, with the accumulation of whole genome and expressed sequences (mRNA and ESTs) in public databases, a significant number of NATs has been identified using computational analysis -22. ThesThe major obstacle in using expressed sequence data for NAT identification is how to determine the correct orientation of the sequences, especially ESTs. Many ESTs were not directionally cloned and even well-known mRNA sequences were registered from both strands of cloned cDNAs or are incorrectly annotated. As done by others ,22,23, wTo identify transcripts that derive from opposite strands of the same locus, we used a modified version of an in-house knowledgebase previously described for humans -28. Thistrans-NATs. Thus, the total number of NATs is likely to be even higher in both genomes. Data presented in Table We first designed software that searched the human and mouse genomes extracting gene information from transcripts mapped onto opposite strands of the same locus. Several parameters were used by the software to identify S-AS pairs, such as: sequence orientation given by the respective GenBank entry; presence and orientation of splice site consensus; and presence of a poly-A tail . We found 3,113 and 2,599 S-AS pairs in human and mouse genomes, respectively, containing at least one full-insert cDNA (sequences annotated as 'mRNA' in GenBank and referred to here as such) in each orientation  heterogeneity, the total number of clusters would be inflated, leading to an erroneous count of S-AS pairs. To evaluate this possibility, we relaxed our clustering parameters, requiring a minimum of 1 base-pair (bp) same strand overlap for clustering. Furthermore, we collapsed into a single cluster all pairs of clusters located in the same strand and less than 30 bp away from each other. Additional data file 3 shows the total number of clusters and S-AS pairs after this new clustering strategy was employed. As expected, both the total number of clusters and S-AS pairs decreased with the new clustering methodology. The total number of clusters decreased by 2% and 1% for human and mouse, respectively, while the total number of S-AS pairs decreased by 0.3% for both human and mouse. Thus, the small difference observed does not affect the conclusions on the genomic organization of S-AS pairs. For all further analyses, we decided to use the original dataset obtained with a more stringent clustering methodology.p = 0.003 for embedded pairs; chi-square = 49, df = 2, p = 2.3 × 10-11 for 5'5' pairs; chi-square = 132, df = 2, p = 2.1 × 10-29 for 3'3' pairs). This peculiar distribution will be further discussed in the light of the expression analyses. Since these intronic NATs have been shown to be over-expressed in prostate tumors [et al. [We further explored the genomic organization of S-AS pairs using the subset of 3,113 human and 2,599 mouse pairs that contained mRNAs in both sense and antisense orientations. The genomic organization of S-AS pairs can be further divided into three subtypes based on their overlapping patterns: head-head (5'5'), tail-tail (3'3') or embedded (one gene contained entirely within the other) pairs . For humans, 66% of all S-AS pairs containing at least one intronless gene are within the 'embedded' category; Sun et al. [et al. [Another interesting observation is the higher frequency of intronless genes within the set of S-AS pairs Table . About hAll these results clearly show that subsets of S-AS pairs have distinct genomic organization, suggesting that they may play different biological roles in mammalian genomes. Below we will discuss these data in a functional/evolutionary context.et al. [et al. [vice versa. Additional data file 5 lists all S-AS pairs found by the three methodologies discussed above.Using our set of human and mouse S-AS pairs, we measured the degree of conservation between S-AS pairs from human and mouse. Since the numbers reported so far are discrepant, ranging from a few hundred ,6 to almet al. , in whic [et al.  the numbet al. [et al. [There is a predominance of 3'3' pairs in all sets of conserved S-AS pairs. For the first strategy solely based on HomoloGene, 67% of all pairs are 3'3' compared to 19% embedded and 14% 5'5'. For the dataset obtained using the strategy from Engstrom et al. , there i [et al.  to take et al. [Large-scale expression profiling tools have been used to discover and analyze the co-expression of S-AS pairs ,23,34. Qet al. ,37 to seDpnII sites. Since these sites are palindromes, we extract, for each one, two virtual tags , both immediately downstream of the restriction site but in opposite orientations . In this way, we could evaluate the expression of transcriptional units present in both strands of DNA. We obtained 5,580,158 and 8,645,994 virtual tags for the human and mouse genomes, respectively. This set of virtual tags was then compared to a list of tags observed in the MPSS libraries. As true for any study using mapped tags, our analysis misses those cases in which a tag maps exactly at an exon/exon border at the cDNA level.We made use of 122 MPSS libraries derived from a variety of human and mouse tissues . Our strategy was based on the generation of virtual tags from each genome by simply searching the respective genome sequence for We first evaluated the number of cDNA-based S-AS pairs -MPSS technique  to conveAmong the 49.5% (91 - 46 = 45) of candidates that were not considered to be validated, we found 25 that were amplified in the GLGI-MPSS experiment but whose exon-intron organization was identical to the sense gene. Although antisense sequences like these have already been observed , we did et al. [Dahary et al.  observedet al. . We decip < 0.0001).If alternative polyadenylation is a significant factor in defining S-AS pairs, we would expect a lower rate of alternative polyadenylation in chromosome X, which has the smallest density of S-AS pairs. Indeed, only 20% of all messages from the X chromosome show at least two polyadenylation variants, compared to 27.5%, on average, for the autosomes  had their 3' ends aligning to stretches of As in the human genome. This motivated us to search for similar cases in the set of cDNA-based S-AS pairs identified in this study. We found that 18% and 26% of all S-AS pairs have at least one gene with its 3' end aligning with a stretch of A's in the human and mouse genomes, respectively. This number is certainly inflated by ESTs since it decreases to 11.7% for human and 12.6% for mouse when only mRNA/mRNA S-AS pairs are considered. Two possibilities could account for this observation. First, a fraction of all antisense transcripts would be artifacts due to genomic priming with contaminant genomic DNA during cDNA library construction. An alternative is the possibility that antisense genes were constructed during evolution by retroposition events. Both possibilities are in agreement with the observation that antisense genes are depleted of introns.An experimental strategy was developed to evaluate the likelihood of genomic priming as a factor generating artifactual antisense cDNAs. A total of 11 mRNA candidates derived from cDNA libraries from fetal liver, colon and lung with a high proportion of sequences that had their 3' ends aligning to stretches of As in the human genome were selected for experimental validation by RT-PCR. cDNA samples used in these experiments were reverse transcribed from fetal liver, colon and lung total RNA treated or not with DNAse. As can be seen in Figure Some other features support the artifactual origin of these antisense transcripts. First, cDNAs containing a stretch of As at their 3' genomic end have much less polyadenylation signals than genes in general (17% compared to 85%). Furthermore, these genes have a much narrower and rarer expression pattern when analyzed by SAGE and MPSS than genes in general (data not shown). These observations suggest that a significant fraction of all antisense genes are actually artifacts, due to genomic priming during library construction.et al. [et al. [bona fide genes [Retroposition generates intronless copies of existing genes through reverse transcription of mature mRNAs followed by integration of the resulting cDNA into the genome . Eventuet al. . Retropoet al. -45. Rece [et al.  found alde genes .et al. [Retrocopies usually have a poly-A tail at their 3' end because of the insertion of this post-transcriptional modification together with the remaining cDNA. Thus, retroposition can explain the high incidence of antisense transcripts with a poly-A tail at their 3' end. To evaluate the contribution of retrocopies to the formation of S-AS pairs we compared the loci identified by Marques et al.  as retroet al. [A critical issue to effectively evaluate the role of antisense transcripts in regulating distinct cellular phenomena is related to the expression pattern of both sense and antisense transcripts belonging to the same S-AS pair. Several reports have been published based on large-scale gene-expression analyses ,23,47,48et al. ,49. To eet al. [p = 5.4 × 10-31). This supports a previous conclusion from Sun et al. [p = 1.2 × 10-6). We observed no statistical difference among the three categories regarding differential expression of both genes in a pair.Figure et al. . The difet al. [cis and trans have been identified , it is in vitro . A few yin vitro  observedn et al.  characteproposed . Howeverp = 2.3 × 10-8 for donor sites; and chi-square = 23, df = 1, p = 1.6 × 10-6 for acceptor sites).We first tested whether the rate of alternative splicing in the sense gene would be affected by the existence of an antisense transcript. It is expected that the effect of S-AS pairing on splicing would be restricted to those exon-intron borders located in the region involved in pairing. We therefore restricted the analysis to those exon-intron borders spanning the region involved in an S-AS pairing. Our strategy was to compare the number of splicing variants for those borders against all other exon-intron borders (those without an antisense transcript) in the same genes. To make the analysis more informative we split the borders into four categories . For both internal donor and acceptor sites, the presence of an antisense transcript slightly increased the rate of alternative splicing  might produce S-AS pairs with different functional implications. The expression and evolutionary conservation analyses presented here, together with evidence from others ,23,47,48This is the deepest survey so far of S-AS pairs in the human and mouse genomes. We made use of all cDNAs available in the public domain together with 122 MPSS libraries for human and mouse. The major findings of the present report include: as many as 10,077 and 8,091 S-AS pairs were identified for human and mouse respectively; using MPSS data, we found 4,308 and 216 new putative S-AS loci in human and mouse, respectively; a small fraction of all S-AS pairs are artifacts caused by genomic priming during cDNA library construction; a significant amount of S-AS pairs is due to retroposition events of one of the genes in the pair; quantitative analyses suggest that the presence of an antisense gene, complementary to an exon-intron border of the sense gene, increases the rate of retention of the respective intron. Furthermore, we propose an evolutionary model in which alternative polyadenylation and retroposition are important forces in the generation of S-AS pairs.Taken together, these results offer, up to now, the vastest catalog of S-AS pairs in human and mouse genomes.We used a modified protocol similar to the one described previously to identify transcription clusters in the human and mouse genomes ,28. BrieCorrect orientation of ESTs was determined by the presence of a poly-A tail (a stretch of 8 As at the 3' end) and/or a splicing donor (GT) and acceptor (AG) sites. All mRNAs were considered in the 'sense' orientation (oriented from 5' end to 3' end). All cDNAs mapped and reliably orientated were assembled into clusters. One cluster contains cDNAs presenting the same orientation and sharing at least one exon-intron boundary or a minimum of 30 nucleotides of overlap (only for those sequences without a common exon/intron organization).DpnII sites and extracting a 13 (human) or 16 (mouse) nucleotide long sequence immediately downstream of the restriction site in both orientations. These 'virtual' tags present only once in the respective genomes were further used and matched against the 'real' tags found in 41 and 81 MPSS libraries for human and mouse, respectively. Only MPSS tags classified as 'reliable' (present in more than one sequencing run) and 'significant' (tags per million >3) were considered as trusted signatures.For the mapping of MPSS data, we first extracted 'virtual' tags for both human and mouse genomes by simply finding all S-AS pairs were identified as those cases in which two clusters, in opposite orientations, overlap at the genome level. For the correct orientation of all mapped cDNAs, we took into consideration several parameters, including: sequence annotation as available in the respective GenBank entry; splice junctions; and poly-A tails and poly-T heads. We excluded from our analyses all cDNAs that presented conflicting orientations as defined by the three criteria above. If only two clusters overlap in the opposite orientation, they were classified as a single bidirectional S-AS pair. If a given cluster overlaps with more than one antisense cluster, they were classified as multiple bidirectional S-AS pairs. S-AS pairs were also classified according to their genomic pattern. Parameters evaluated included: pattern of S-AS overlap ; spanning of introns by the components of a pair as defined by their alignment onto the genome; and chromosome localization and relative orientation within the S-AS pairs .et al. [We used three strategies to evaluate the degree of conservation between human and mouse S-AS pairs. First, all pairs were searched against the dataset from HomoloGene  and thoset al. . We usedDpnII restriction sites of each mRNA sequence). To accurately represent the 3' end of a transcript, only mRNA sequences containing a poly-A tail were used. All tags mapped to two or more different genes were excluded and the frequencies of different tags for the same gene  were summed. MPSS tags were normalized to counts-per-million and the expression data were cross-linked to genomic positions by the extraction of virtual tags for both the human and mouse genomes. Only tags showing 100% identity with a genomic locus were used in the analyses.We evaluated the expression pattern of S-AS pairs at the whole genome level based on their expression profiles obtained from MPSS libraries (available at ). The prThe classification of the expression pattern of S-AS pairs was done using those tags with ≥3 tags per million across all MPSS libraries. To evaluate the co-expression of all S-AS pairs, both genes in a pair had to be co-expressed in at least 04 libraries. If both genes in a pair were co-expressed in less than four libraries or they were independently expressed in different libraries, the pair was classified as 'single-gene expression'. The remaining S-AS pairs were classified as 'no-expression'.DpnII sites in the human and mouse genomes were identified and for each site two 'virtual' MPSS tags were extracted from both DNA strands in the correct orientation. All 'virtual' MPSS tags mapped in the opposite strand of known mRNAs in both genomes were identified. Those mRNAs belonging to an S-AS pair previously identified were excluded. Those antisense MPSS tags mapped just once in the respective genome and present in at least one MPSS library were identified and submitted to experimental validation.All A random distribution of S-AS pairs was obtained by re-indexing the coordinates of one gene in all the pairs 1,000 times. This was done by randomly selecting a genomic coordinate for the start of mapping of a given gene. All the remaining exon-intron borders were then re-indexed based on this initial coordinate. The relative organization of both genes in all random S-AS pairs was stored and frequencies for each category were calculated. Those frequencies were used as the expectation for chi-square tests of the null hypothesis.Using the database mentioned earlier and described elsewhere -28 we id2, 83 μM dNTPs, 2.3 ng/μl antisense primer, 2.3 ng/μl sense primer, 1.5 U of Taq Platinum DNA polymerase  and 0.5-0.8 μl of the same cDNA source used for MPSS library construction. PCR conditions used for amplification were 94°C for 2 minutes, followed by 30 cycles at 94°C for 30 s, 64°C for 30 s, and 72°C for 35 s. Reactions were kept at 72°C for 5 minutes after the last cycle. The amplified products were ethanol precipitated and cloned into the pGEM®-T Easy vector . Twelve colonies for each GLGI-MPSS fragment were screened by PCR using pGEM universal primers and positive colonies were sequenced using Big-Dye Terminator  and an ABI3100 sequencer (Applied Biosystems).MPSS tags corresponding to antisense transcripts were converted into their corresponding 3' cDNA fragments using GLGI-MPSS . AntisenDNAse I  and were reverse transcribed using oligo(dT)12-18, random primers and SuperScript II (Invitrogen), following the manufacturers' instructions. After synthesis, the resulting cDNA was subjected to RNase H treatment. The absence of genomic DNA contamination was evaluated for each preparation. DNA-free total RNA was subjected to PCR amplification using primers within intronic sequences flanking exon 12 of the hMLH-1 gene . All PCR amplifications were carried out using 2 μl of cDNA as a template to the final volume of 25 μl and 1× buffer, 1.5 mM MgCl2, 0.2 mM dNTP, 0.2 μM of each specific primer and 0.025 U/μl of Taq DNA polymerase . The following cycling protocol was used: initial denaturation of 94°C for 4 minutes; 94°C for 30 s; 55°C for 45 s; 72°C for 1 minute for 35 cycles; along with a final extension at 72°C for 7 minutes. All PCR products were resolved on 8% polyacrylamide gels and sequenced as described above to verify amplification specificity.Total RNA derived from fetal liver, colon and lung was purchased from Clontech laboratories . For cDNA synthesis, 2 μg of total RNA were treated (or not) with 100 units of SuperScript II (Invitrogen) and 0.9 μM of a primer complementary to the antisense transcript. PCR amplifications were performed using 1 μl of the first-strand cDNA as a template in a final volume of 25 μl and 1× buffer, 1.5 mM MgCl2, 0.1 mM dNTP, 0.4 μM of gene specific primers and 1 U of Platinum Taq DNA polymerase (Invitrogen). The following cycling conditions were used for amplification: initial denaturation of 95°C for 2 minutes; 94°C for 40 s; reaction-specific annealing temperature for 40 s and 72°C for 1 minute for 35 cycles; followed by a final extension step at 72°C for 7 minutes. All PCR products were resolved on 8% polyacrylamide gels. Controls for the absence of self-priming during cDNA synthesis were done with reverse transcriptase in the absence of primers, and controls for the absence of DNA were done by incubation with primers but with no reverse transcriptase.In the strand-specific RT-PCR, orientation of the transcript is accessed by restricting which gene-specific primer is present during first-strand cDNA synthesis. For each candidate, 1 μg of total RNA was treated with Promega RQ1 RNAse-free DNAse and tested for remaining DNA contamination as described above. First-strand cDNA synthesis was carried out at 50°C for 2 h using 200 U of To make our dataset fully accessible to the community we have set up a worldwide web portal  containiSERF2 and HYPK) with a NAT located between them. Additional file The following additional data are available with the online version of this paper. Additional data file Representative GenBank entries for all S-AS pairs in both human and mouse.Click here for fileTotal number of S-AS pairs by chromosome for both human and mouse.Click here for fileNumber of clusters and S-AS pairs when a less stringent clustering methodology is applied.Click here for fileAll possible genomic organizations of S-AS pairs.Click here for fileAll S-AS pairs conserved between human and mouse using the three strategies described in the text.Click here for fileThe fraction of S-AS pairs conserved between human and mouse that are classified as 'Fully intronic' and the fraction of conserved S-AS pairs that contain at least one intronless gene.Click here for fileAll MPSS libraries used in this study.Click here for fileThe number of cDNA-based pairs that were further confirmed by the MPSS data.Click here for fileSERF2 and HYPK) with a NAT located between them.Chimeric transcripts joining two adjacent genes (Click here for fileAll cases of chimeric transcripts identified in our dataset.Click here for file


SMARCA2). We have examined the association of this polymorphism with schizophrenia in a family-based and case/control study. SMARCA2 was chosen as a candidate gene because of its specific role in developmental pathways, its high expression level in the brain and some evidence of its association with schizophrenia spectrum disorder from genome-wide linkage analysis.Brahma (BRM) is a key component of the multisubunit SWI/SNF complex, a complex which uses the energy of ATP hydrolysis to remodel chromatin. BRM contains an N-terminal polyglutamine domain, encoded by a polymorphic trinucleotide (CAA/CAG) repeat, the only known polymorphism in the coding region of the gene  and unaffected controls (n = 273) in each of three Caucasian populations studied: French Canadian, Tunisian and other Caucasians of European origin.SMARCA2 and schizophrenia.Results from our family-based and case-control association study suggest that there is no association between the trinucleotide repeat polymorphism within  The SWI/SNF complex is a 2MDa, multi-subunit complex that uses the energy of ATP hydrolysis to disrupt nucleosome structure thereby increasing accessibility of transcription factors to their specific sites on DNA and histones. It is well conserved, with homologous complexes isolated from yeast, Xenopus, Drosophila and mammals . SWI/SNFSMARCA2 gene. It is a member of the large, diverse SMARC  family. The only polymorphism within the encoding region of this gene is a stable, polymorphic trinucleotide repeat. We have carried out a case/control and family-based study to examine the association of this polymorphism with schizophrenia (SCZ).BRM is encoded by the SMARCA2 as a candidate gene for SCZ, is plausible for several reasons. First, genome-wide expression analysis of a post-mortem section of the dorsolateral prefrontal cortex of schizophrenia patients showed that SMARCA2  was upregulated 1.42-fold relative to non-affected controls [SMARCA2 has not been confirmed in other studies, several groups have demonstrated significant differences in mRNA levels in schizophrenic patients relative to controls. Gene expression profiles using post-mortem brain tissue  showed that 14% of the genes were up-regulated while 9% were down-regulated more than two-fold in the SCZ group relative to controls [Investigating controls . Even thcontrols . These rcontrols ,9.A second reason why an association between BRM and schizophrenia is plausible is that BRM appears to play an important role in cell development in general, and neuronal differentiation in particular and SCZ is believed to be a neurodevelopmental disorder . Unlike SMARCA2 is located on chromosome 9p22.3. Genome scan meta analysis indicated that region 9p22.3-21.1 demonstrated significant linkage (p < 0.01) with a schizoaffective-bipolar and bipolar-I disease model [Finally studying BRM as a candidate gene is supported by some linkage studies. se model .Saccharomyces cerevisiae Swi2/Snf2 proteins  being a trinucleotide repeat (CAA/CAG) polymorphism. The most commonly occurring allele, corresponding to 32 trinucleotide repeats  was designated the "0" allele. The two next most commonly occurring alleles were designated -1 (178 bps) and +1 (184 bps). Rare alleles -7, -5, +2, +3, and +4, having an allele frequency less than 0.01, were also obtained. Due to the problems that can arise in the cross-tabulation, since these cells had less than 5 cases each, the alleles were grouped for the statistical analyses- alleles -7, and -5 were grouped with the -1 allele and +2, +3, and +4 were grouped with the +1 allele.BRM encapsulates several distinct domains including a polyglutamine domain, the DNA-dependant helicase domain and the bromodomain. The N-terminal polyglutamine domain is well conserved in mammals with glutamine-rich regions also present in the homologous Drosophila Brahma and s Figure . BRM is p = 0.034) was observed. However this effect was not significant when corrected for multiple testing.Case/control analysis was carried out to compare the allele and genotype frequency of the trinucleotide repeat polymorphism within three Caucasian populations: French Canadian, Tunisian and other Caucasians of European origin. Similar allele and genotype distributions were observed between affected cases and unaffected controls in each of these three populations Tables  and 4. TSMARCA2 and schizophrenia. Although all subjects included in the study were carefully diagnosed according to DSM criteria, as having schizophrenia or schizoaffective disorder, one cannot exclude the phenotypic diversity that is inherent to the disorder. For example, within our French Canadian group of affected subjects, 70.7% were diagnosed with paranoid schizophrenia, 5.05% with the disorganized type, 2.02% with schizoaffective disorder and 22.22% with undifferentiated schizophrenia. There has been considerable discussion recently that in studying the genetics of schizophrenia and complex psychiatric disorders, in general, that it is necessary to examine suitable endophenotypes within the disorder rather than to examine the disorder as a whole [SMARCA2 with specific endophenotypes resulting from neurodevelopmental deficits, given the importance of BRM in development and neural development in particular. Also given the minor effect detected with the family-based association study, it may be important to re-examine this polymorphism with a larger sample size.These results suggest that there is no association between the trinucleotide repeat polymorphism within  a whole -19. It rQ23-P- Q3 -P2 -Q- P-Q-. The sequence of the -1 allele was -Q22 -P- Q3 -P2 -Q- P -Q- while the sequence of the +1 allele was -Q24 -P- Q3 -P2 -Q- P -Q-. It therefore appears that the terminal CAG repeat in the first block of CAA/CAG repeats is the polymorphic site, at least in the individuals studied. This is consistent with results obtained in examining the different alleles at this locus in an Indian population [This study also offers a step forward in understanding the polyglutamine domain within BRM. Although BRM has been shown to be important in transcriptional regulation, little is known about the role of the polyglutamine domain. In the populations we have studied, we have not observed an expansion of the trinucleotide repeat region, even though the number of glutamines (32) is close to the pathogenic threshold of ~35–40 glutamines . The tripulation . FurtherSMARCA2 and schizophrenia.Results from our family-based and case-control association study suggest that there is no association between the trinucleotide repeat polymorphism within Subjects diagnosed with schizophrenia or schizoaffective disorder, their families and unrelated healthy controls were recruited from three populations: Tunisian, French Canadian and other Caucasians of European origin. Patients were diagnosed using DSM-III-R or DSM-IV criteria on the basis of the Diagnostic Interview for Genetic Studies (DIGS) and complementary data from the medical files. Most of these subjects had previously participated in a pharmacogenetic study with a detailed evaluation of therapeutic response to medication . For theCase/control analysis was conducted exclusively with subjects from Montreal, Ottawa and Tunisia since unaffected control subjects were not available for analysis from Hungary and France. This step was undertaken to minimize any effects arising due to population stratification. Unrelated cases n = 289) and controls (n = 273) were used for the case-control analysis. The cases included  to ensure that the individual did not have an ongoing or previous psychiatric illness. Written, informed consent was obtained from each patient and non-affected subject prior to beginning the study. The research protocol and the consent forms were approved by the Ethics Committee of each Institutional Review Board.SMARCA2 was amplified by polymerase chain reaction (PCR) using the following primers: forward-5'TGCAGTCCAGGGGAAAAGGACGTT3', reverse-5'CCCGGGCCAGATGGTCTGTTGTAG3'. The forward primer was fluorescent labeled with 6-FAM™ (Applied Biosystems). A 15 μl PCR reaction, containing 10 ng DNA, 2.5 mM MgCl2, 0.25 mM of each dNTP, 0.33 μM of each primer, and 0.6 units Taq Polymerase was carried out. After initial denaturation at 94°C for 5 minutes, 35 cycles were carried out at 94°C for 1 min, 61.3°C for 1 min, 72°C for 1 min, followed by a final extension at 72°C for 30 min.Genomic DNA was extracted from peripheral blood using standard procedures. The trinucleotide repeat within ® 3100 Genetic Analyzer and GeneScan v3.7. The sizes of the DNA fragments were obtained in comparison to the DNA standard using Genotyper® Software v3.7. The most frequently occurring allele, corresponding to a size of 181 bp (obtained using Genotyper®) was designated "0". The sizes of each of the respective alleles increased or decreased proportionately by 3 bps i.e. alleles -1, and +1 had sizes of 178 and 184 bps respectively.After verification of the PCR amplification by agarose gel electrophoresis, 0.5 μls of each sample was mixed with 0.3 μls size standard (GeneScan™ 500 LIZ™) and 9.2 μls deionized formamide (Applied Biosystems), denatured at 95°C for 5 minutes, and placed on ice. Each 96-well plate was analyzed using the ABI PRISMχ2) statistic in Statistica®. Statistical significance was established at p < 0.05.The Transmission Disequilibrium Test  was condBRM – BrahmaDIGS – Diagnostic Interview for Genetic StudiesFBAT – Family-Based Association TestsPCR – polymerase chain reactionSCID-NP – Structured Clinical Interview for DSM-IV, Non-patient editionSCZ – schizophreniaSMARC – SWI/SNF-related, matrix-associated, actin-dependant regulator of chromatinSS, LX, FF performed the data analysis. SS drafted the manuscript. CB, KT, ZD, AL, SL, MK, GR and RJ were involved in the study design and provided clinical support. RJ was responsible for the conception of the study, supervision of the research project and drafting of the manuscript.


Helicobacter pylori colonization is associated with the development of distal gastric cancer (GC). The host response to H. pylori has been related to genetic polymorphisms that influence both innate and adaptive immune responses.The intensity of the inflammation induced by TLR4 Asp299Gly, TLR4 Thr399Ile and IL-8-251 A/T polymorphisms had any influence in the development of distal GC in a Mexican population.Our aim was to investigate whether the presence of the H. pylori in the control population was defined by positive results of at least two of four diagnostic tests: serology, histology, rapid urease test and culture. Human DNA was purified and genotyped for TLR4 Asp299Gly polymorphism by pyrosequencing, for TLR4 Thr399Ile by PCR-RFLP and for IL8-251 by the amplification refractory mutation system (ARMS)-PCR.We studied 337 patients that were divided in two groups: 78 patients with histologically confirmed distal GC and 259 non-cancer controls. The presence of H-W = 0.58 for IL8-251, 0.42 for TLR4 Asp299Gly and 0.17 for TLR4 Thr399Ile). The frequencies of mutated alleles (homozygous plus heterozygous) were compared between cases and controls. We found no significant difference for TLR4- Asp299Gly [the 7.7% of distal GC patients and 7.7 % non-cancer controls (p = 0.82)] and for TLR4 Thr399Ile [the 1.3% of GC patients and the 5% of the control population (p = 0.2)]. In contrast, for IL-8-251 A/T, 80.77% of the GC patients and 66.4% in the control group age and gender matched had at least one copy of mutated allele  (p = 0.023).The non-cancer control group was found to be in Hardy-Weinberg equilibrium at the polymorphic loci studied (chi-square IL8-251*A allele could be related to the development of distal gastric cancer in this Mexican population.This study showed that the  Helicobacter pylori almost exclusively colonize the mucous layer of the human stomach and reside in the host for years, decades or maybe lifelong. In approximately 10% of cases, the organism is associated with diverse clinical outcomes, including non-ulcer dyspepsia, peptic ulcer disease, and distal gastric cancer [Both host and environmental factors have been associated with this clinical diversity. Host factors are mainly related to the recognition of the bacteria by the immune system and variation in the level of cytokine response ,3.H. pylori [TLR4 gene, Asp-299→Gly and Thr-399→Ile transitions, have been shown to lead to hyporesponsiveness to LPS, reduced epithelial TLR4 density and reduced inflammatory cytokine response to LPS [(IL)-1B, TNF, IL-10 and IL-8 [Recognition of pathogens is mediated by a set of germline-encoded receptors that are referred to as pattern-recognition receptors (PRRs). These receptors recognize conserved molecular patterns, which are shared by large groups of microorganisms. An important PRR is Toll-like receptor 4 (TLR4), a transmembrane receptor that recognizes a range of ligands, including lipopolysaccharide (LPS), which is found in the cell wall of Gram-negative bacteria like . pylori . Two sinand IL-8 -7.IL-8 cytokine functions as a potent chemoattractant for neutrophils and lymphocytes. It has been reported that IL-8 levels increased 10-fold in gastric cancer specimens when compared with normal gastric tissues. A well-characterized SNP T/A in the -251 locus has been described and associated with higher IL-8 production and several diseases .TLR4 Asp299Gly, TLR4 Thr399Ile and the IL8-251 A/T polymorphisms are associated with distal gastric cancer in a well-defined Mexican population.Our aim was to determine whether the We studied 78 unrelated patients with histologically confirmed distal GC . We also studied 259 patients with no histological evidence of GC . This population has been described previously [From all control patients, eight biopsy specimens were obtained for histological evaluation, two from the lesser curvature, two from the greater curvature, two from the incisura angularis, and two from the prepyloric region. From patients with distal GC, at least 8 biopsies were obtained from the tumor for histological evaluation. Biopsies from cases and controls were fixed, paraffin embedded and stained with hematoxilin-eosin. An experienced pathologist examined all the histological slides.H. pylori status of the control population was determined by histology, the rapid urease test (RUT), serology and culture. For the RUT, an antral biopsy was analyzed by a validated non-commercial test [The ial test . The bioial test .Culture was performed on two corpus and two antrum biopsies by standard methods. Biopsies were placed in 10% blood agar  and incubated at 37°C for at least one week under microaerobic conditions. Strains were identified by Gram staining, oxidase, catalase and urease tests.H. pylori infected when at least two of the diagnostic tests were positive.Control patients were considered H. pylori infection in gastric cancer patients was determined by the result of histology only.The prevalence of IL8-251 polymorphism as described previously [Genomic DNA was extracted from peripheral blood by the phenol-chloroform-isoamilic alcohol and precipitation with ethanol method. An amplification refractory mutation system (ARMS)-PCR method was used to genotype the eviously .TLR4 Thr399Ile polymorphism was determined by PCR-RFLP [TLR4 Asp299Gly polymorphism was determined by a pyrosequencing method designed for this study. Initially, a PCR was performed with the primers F-GAT TAG CAT ACT TAG ACT ACT ACC TCC ATG and R-CCC TTT CAA TAG TCA CAC TCA CCA GG with the reverse primer biotinylated. The PCR conditions were: 94°C for 5 min and 40 cycles of 94°C, 59°C and 72°C for 30 s each, and a final extension at 72°C for 5 min. For pyrosequencing, biotinylated PCR templates were immobilized on streptavidin-coated paramagnetic Sepahrose beads in binding buffer. The bead-template complexes were washed by submersion in 70% alcohol and 0.5 M NaOH and then added to 45 μl of annealing buffer containing 15 pmol of the sequencing primer 5'-CAT ACT TAG ACT ACT ACC TC 3'. Annealing took place at 80°C for 2 min. Real-time pyrosequencing was performed in an automated 96-well pyrosequencer PSQ SNP 96  using enzymes and substrates recommended by the manufacturer.The PCR-RFLP  and the t test, chi-square or Fisher exact test, two tailed. A probability (p) value < 0.05 was considered as statistically significant. Odds ratios (OR) with 95% confidence intervals (CIs) were computed using the Epi-Info 2000 software . Three analyses were performed, one in which all cases and controls were included, a second in which cases were compared only with H. pylori + controls and a third analysis in which the population-based controls were matched by 5-year age group and gender with the cancer cases.Hardy-Weinberg equilibrium of alleles at individual loci was assessed by chi-square statistics. Statistically significant differences were determined by Student's The distribution of genotypes according to histological findings is shown on Table IL8-251, 0.42 for TLR4 Asp299Gly and 0.17 for the TLR4 Thr399Ile). Among the control group, the 80% had chronic gastritis , for TLR4 Asp299Gly  and TLR4 Thr399Ile   for GC cases and all gastritis plus normal controls. We found an association of the IL8-251 *A allele and the development of GC  .We next compared the carriage of the H. pylori+ controls we found a similar value .When we compared the gastric cancer cases with the Next, we compared the frequency in the gastric cancer group withage and gender matched controls and we found a stronger association .TLR4 Asp299Gly*2 and TLR4 Thr399Ile*2 hyporeactive alleles.We did not find any association for H. pylori has been attributed to several factors. The genetically regulated immune response seems to play a crucial role in determining the intensity of damage to the host [The diversity in response to the infection by the host -3.H. pylori infection. An A/T polymorphism at the position -251 in the gene has been associated to a higher activity of this interleukin and recently, this allele has been related in two studies to the development of gastric cancer in Japanese population [IL8-251 AA genotype held a higher risk of atrophic gastritis  and gastric cancer  compared with the TT genotype. They studied 252 healthy controls, 215 individuals with atrophic gastritis, and 396 patients with gastric cancer. The AA and AT genotypes were significantly associated with higher levels of IL8 protein, more severe neutrophil infiltration compared with the TT genotype. In the other study, the IL-8 -251*A allele was associated to a higher risk of gastric cancer . Our results showed no association between higher rates of GC and the AA genotype, but a positive association was found for the AT genotype and the carriage of at least one copy of the *A allele (homozygous plus heterozygous). It is remarkable that the ORs value observed in our study (2.12) was quite similar to that observed in the Japanese population (2.1).Several authors have described the increased production of IL-8 during pulation ,11. In oIL-1B-31 C/T polymorphism has been associated with the development of gastric cancer and a geographical-ethnic difference has been described for this locus, with a stronger association of the polymorphism in occidental than in oriental population. The results for the IL-8-251 A/T polymorphism correlate with previous data presented for Japanese population, showing no trend in geographical or ethnic variation.The TLR4 polymorphisms have shown reduced levels of circulating inflammatory cytokines, an increased risk of acute bacterial infections and a trend towards increased mortality. Other recent studies support the hypothesis that the low-functioning TLR4 polymorphisms may result in a reduced inflammatory response associated with a low-damaging infection that will promote a persistent infection [TLR4 Asp299Gly and Thr399Ile polymorphisms have been reported in linkage disequilibrium (LD) [It is currently unresolved whether a hyporesponsive LPS signaling pathway is beneficial or detrimental to the host. Individuals with the hyporeactive nfection -20. In tium (LD) ,22, but IL8-251 is a biologically plausible disease-modifying polymorphism. If confirmed in multiple populations, as it has been in Japanese, and now Mexican groups, this polymorphic site, in conjunction with others, could be used to identify patients at greater risk for GC, who may need closer monitoring.In conclusion, our preliminary results suggest that The author(s) declare that they have no competing interests.EGG designed and coordinated the study, optimized PCR conditions, carried out molecular analysis and drafted the manuscript. SM performed PCRs analysis, JPFG performed histological examination and interpretation of results, FJBP and HJMG recruited patients and carried out endoscopies and gastric biopsies, GIPP conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Down syndrome (DS) is caused by trisomy 21 (+21), but the aberrations in gene expression resulting from this chromosomal aneuploidy are not yet completely understood.We used oligonucleotide microarrays to survey mRNA expression in early- and late-passage control and +21 fibroblasts and mid-gestation fetal hearts. We supplemented this analysis with northern blotting, western blotting, real-time RT-PCR, and immunohistochemistry.MX1 was strongly over-expressed (mean 16-fold) in senescent +21 fibroblasts, a result verified by northern and western blotting. MX1 is an interferon target gene, and its mRNA was induced by interferons present in +21 fibroblast conditioned medium, suggesting an autocrine loop for its over-expression. By immunohistochemistry the p78MX1 protein was induced in lesional tissue of alopecia areata, an autoimmune disorder associated with DS. We found strong over-expression of the purine biosynthesis gene GART (mean 3-fold) in fetal hearts with +21 and verified this result by northern blotting and real-time RT-PCR.We found chromosome 21 genes consistently over-represented among the genes over-expressed in the +21 samples. However, these sets of over-expressed genes differed across the three cell/tissue types. The chromosome 21 gene Different subsets of chromosome 21 genes are over-expressed in different cell types with +21, and for some genes this over-expression is non-linear (>1.5X). Hyperactive interferon signaling is a candidate pathway for cell senescence and autoimmune disorders in DS, and abnormal purine metabolism should be investigated for a potential role in cardiac defects. Trisomy 21 (+21) is the most common human chromosomal aneuploidy at term, and the only one with long-term viability. This syndrome has attracted intense research interest as a prevalent cause of mental retardation, and adults with DS develop pathological and neuropsychiatric aspects of Alzheimer's disease as early as age 40. DS is also associated with phenotypes outside of the central nervous system. Among these are cardiac septal defects, autoimmune diseases, and signs of premature cellular senescence including skin wrinkling. A unifying explanation for the diverse features of DS is not apparent, although they are presumably due to abnormal expression of specific sets of critical genes, both on chromosome 21 and, through gene-gene interactions, elsewhere in the genome.Attempts to define a DS "critical region" on chromosome 21 by correlating clinical phenotypes with the locations of sub-chromosomal segments duplicated in individuals with chromosome 21 unbalanced translocations have been somewhat successful, but the critical region for the complete syndrome still encompasses most of the q-arm of this chromosome . SimplisFive normal and six DS skin fibroblasts were obtained from the American Type Culture Collection , from Coriell Laboratories , or as de-identified samples from the cytogenetics laboratory, stored by the Columbia University Pathology Department Tissue Bank. All of the cell were maintained and passaged at a ratio of 1:4 in DMEM  containing 10% FBS . Cell senescence was scored by activity of acid β-galactosidase every 3 to 5 generations. Tissue from fetal hearts at 15 to 23 weeks gestation, from cases of +21 and controls, was obtained as de-identified cryopreserved samples from the Columbia University Pathology Department Tissue Bank. Total RNA was prepared by a two-step procedure using Trizol™ reagent (Invitrogen), followed by RNeasy  purification. This work was carried out in compliance with the Helsinki Declaration: since the human fibroblast and heart tissues were de-identified samples, this study was not considered human subjects research by the Columbia University Internal Review Board.Total RNA was resolved on formaldehyde-containing agarose gels and transferred to Nytran membranes . The probes for northern blotting were partial cDNAs prepared from RT-PCR using gene-specific primers. Hybridization was in ULTRAhyb buffer (Ambion) at 42°C degrees. Probes were stripped by boiling the membranes in 0.1 % SDS/0.1 × SSC for 2 minutes, or were allowed to decay without stripping.GART, sense primer 5'-CCATAGCTTTCCTCCAGCAG-3' and anti-sense primer 5'-AGTTCCAACGCCATCTGTTC-3'; HPRT (housekeeping gene control), sense primer 5'-TTGCTCGAGATGTGATGAAGGA-3' and anti-sense primer 5'-TTCCAGTTAAAGTGGAGAGATCA-3'.Total RNA, 2 μg, was reverse transcribed with oligo-dT primers using Superscript version II reverse transcriptase (Invitrogen). Quantitative real-time RT-PCR was performed with the Master SYBR Green I system  in a LightCycler apparatus (Roche) with an initial denaturation at 95°C for 30 seconds followed by multiple cycles of denaturation at 95°C for 5 seconds, primer annealing at 62°C for 5 seconds, and extension at 72°C for 12 seconds. LightCycler Software 4.0 (Roche) was used for data analysis, with mRNA content determined from inflection points of SYBR Green fluorescence plotted as a function of cycle number. The following oligonucleotide primers were used for the PCR reactions: MX1 or α-actin  in a solution containing 0.1% BSA, 0.1% Tween-20, and 3% dry milk overnight at 4°C. After washing, the signal was amplified using a peroxidase-conjugated goat anti-mouse IgG  and was visualized using a commercial ECL plus detection system (Amersham Pharmarcia Biotech).After boiling for 10 minutes in a denaturing solution containing 12 mM Tri pH6.8, 5% glycerol, 0.4% SDS, 3 mM 2-mercaptomethanol, and 0.02% bromophenol blue, 30 μg of total protein lysates from +21 and control fibroblasts were subjected to electrophoresis on SDS-denaturating 4–20 % polyacylamide gels (Invitrogen). After transferring to the nitrocellulose membrane and blocking by 5% milk, the membrane was hybridized with mouse antibodies against p78HGU95A GeneChips were purchased from Affymetrix . Double stranded cDNA was generated from 5 μg of total RNA using a poly-dT oligonucleotide that contained a T7 RNA polymerase initiation site and the Superscript Choice System Kit (Invitrogen). The cDNA was phenol/chloroform extracted, and biotinylated cRNA was generated by in vitro transcription using the Bio Array High Yield RNA Transcript Labeling System . The cRNA was purified using RNeasy. cRNA was fragmented according to the Affymetrix protocol, and 15 μg of biotinylated cRNA were hybridized to U95A microarrays (Affymetrix). After scanning, the expression values for each gene were determined using Affymetrix GeneChip software v. 5.0. For each sample, the signals for each gene (probe set) were normalized to the values for the entire microarray, using GeneSpring software . Statistical analyses and automated clustering of the microarray data were done using the Filter Genes and Gene Tree functions of GeneSpring. For supervised analysis, we used ANOVA to identify all genes that differed at p < .05 and, in a separate analysis, at p < .01, in a given cell type  as a function of the presence or absence of +21. The resulting small sets of genes were then further filtered for data quality by requiring Affymetrix 'presence calls' (high-reliability hybridization data) in at least 3 samples, and were filtered a third time for magnitude of the differences in hybridization signals by requiring at least 3 samples with signals >1.2-fold above the experiment mean. We did not correct statistically for multiple comparisons  since doing so would reduce the statistical power far below the level needed to detect changes in the 1.5-fold range. We used the Gene Tree function to group the differentially expressed genes according to the extent of similarity in their patterns of expression across the samples, thereby separating the genes into over-expressed and under-expressed clades. Fold over-expression of each gene was calculated as the mean of the normalized signals for the +21 samples divided by the mean of the control samples.MX1 at room temperature overnight. After washing in TBS-T, slides were incubated with biotinylated secondary anti-mouse IgG antibody  for 30–40 minutes at room temperature. Antigen-antibody complexes were developed using the Vectastain ABC kit (Vector Laboratories) and a chromogenic substrate, diaminobezidine (Dako). Sections were lightly counterstained with hematoxylin. For assessing the effects of interferon in +21 conditioned medium, we used a goat anti-beta-interferon antiserum (Sigma-Aldrich).Sections of archival formaldhehyde-fixed scalp biopsies were deparaffinized in xylene and hydrated through graded ethanols. Antigen retrieval was carried out in 1 mM EDTA buffer by boiling the slides in a microwave oven for 8 minutes at the maximum power and, sequentially, boiling for 15 minutes at a reduced power. Endogenous biotin was blocked by two steps of incubation for 10–20 minutes with egg white and 5% skim milk in 1× Tris-buffered saline (TBS) containing 0.5% BSA and 0.1 % NaN3, respectively. Between the two blocking step for biotin, slides were treated with 0.3% hydrogen peroxide in 0.1% NaN3 to block endogenous peroxidase activity. Slides were washed three times in 1× TBS buffer containing 0.1% Tween-20 (TBS-T) and then incubated with 5% of the blocking serum for 30 minutes in a humidified chamber. After 3 TBS-T washes, slides were hybridized with a monoclonal antibody against p78For mRNA expression profiling we used Affymetrix U95A GeneChips, which query ~10,000 human genes , including 108 unique genes (143 probe sets) from chromosome 21. Data obtained using these microarrays in our laboratory have previously shown highly reliable correlations with independent measures of gene expression, including northern blotting . We analSeveral noteworthy features were seen in the expression data from the early passage cells. First, genes on chromosome 21 were strikingly over-represented, relative to those on other chromosomes, in the 'high in +21' set. The proportion of chromosome 21 genes was 6/14 (43%) by ANOVA at p < .01, Table MX1, was over-expressed far in excess of the 1.5-fold change expected from chromosome dosage in the +21 fibroblasts  ANOVA test at p < .01 in these senescing cells, and this gene,  .05 see . The oveing Fig. . In normnes Fig. . Accordiable see . While wMX1 mRNA (GeneChip data) vs. the percentage of acid beta-galactosidase-positive cells was 0.63 for normal fibroblasts and 0.72 for the +21 fibroblasts. Thus, MX1 expression was correlated to cell senescence in both types of fibroblasts, albeit with a stronger positive correlation in +21 compared to control, and with a substantially greater magnitude of the increased expression in the +21 cells. In addition, western blotting of total cellular protein indicated that the excess of MX1 mRNA in these cells led to the expected excess production of p78MX1 protein, and we observed a good correlation between MX1 mRNA and p78MX1 protein . Although IFI27 is not on chromosome 21, the receptors for interferon alpha and beta are encoded by genes on chromosome 21. While we did not detect increased mRNA for these genes in our survey, fibroblasts and other cells with +21 are known to express an increased density of cell surface interferon receptors at the protein level, and to be hyper-responsive to signaling by exogenous interferon [MX1 mRNA and protein in +21 cells. Indeed, in early work the protein product of the MX1 gene, p78MX1, was found over-expressed in two lines of +21 fibroblasts, compared to control fibroblasts, after these cells were exposed to exogenous interferon [MX1 in senescing +21 fibroblasts, and we therefore wished to test whether this increased expression might be maintained by autocrine interferon signaling. Consistent with this hypothesis, conditioned medium from +21 fibroblast cultures induced the expression of MX1 mRNA in control fibroblasts, and this induction was attenuated by the addition of anti-interferon-beta antibodies to the cell cultures . In both of two cases of sporadic alopecia areata that we examined there was high expression both in endothelial cells and in a subset of the infiltrating lymphocytes and in epithelial cells (outer root sheath cells and matrix cells) of the inflamed hair follicles, with no immunoreactivity in the adjacent uninvolved hair follicles , which acts at several steps of purine biosynthesis [SH3BGR, encoding a coiled-coil and SH3 domain-containing protein, were modestly over-expressed on average in the +21 hearts  over-expression of this gene, and the modest over-expression of SH3BGR mRNA, by northern blotting and Phosphorimaging in 3 of the hearts with +21 for which ample RNA remained after the GeneChip experiment, compared to 4 control hearts closely matched for gestational age  in 4 of the +21 fetal hearts compared to 6 of the control fetal hearts from our GeneChip series  also revealed a systematic over-expression of chromosome 21-linked genes in the +21 cases. Using ANOVA at a cutoff of p < .01, among 37 genes in the 'high in +21' category, 7 (19%) mapped to chromosome 21; while no chromosome 21-linked genes were in the under-expressed category Fig. . Similarynthesis . In addiage Fig. . This reies Fig. .SH3BGR, which was modestly (i.e. linearly) over-expressed in the hearts and early passage fibroblasts with +21, and MX2, which was modestly over-expressed in both the hearts and the late passage fibroblasts. The limited overlaps between the gene lists from the supplemental tables are highlighted by the Venn diagram in Figure As seen by comparing Tables MX1 gene is strongly (non-linearly) over-expressed in senescing +21 fibroblasts, and that the GART gene, encoding a multifunctional enzyme in de novo purine biosynthesis, is non-linearly over-expressed in mid-gestation fetal hearts with +21. For MX1, non-linear over-expression can be explained at least in part by the existence of more than one interferon pathway component encoded on chromosome 21. We have also shown evidence for induction of the MX1 gene product, p78MX1, in lesional hair follicle tissue of a sporadic case of alopecia areata, an autoimmune disorder that has an elevated frequency in DS. In terms of these genes in the prior literature, Gart was found slightly up-regulated in heart, kidney, and muscle of segmental trisomy 16 mice at day 30 by 1.54, 1.68, and 1.83, respectively [Mx1 and Mx2 genes appeared among the limited set of chromosome 16-linked genes (13 genes) found to have >2-fold over-expression in several adult tissues at postnatal day 10 and at 11 months in the segmental trisomy 16 mice [A number of screens for altered gene expression in DS cells and tissues, and in the segmental trisomy 16 mouse models of DS, have been published over the past several years. Analysis of mRNAs in brain is clearly of major interest, but several prominent aspects of the DS phenotype involve non-CNS tissues. Our analysis of gene expression in fibroblasts and fetal hearts with trisomy 21 leads us to a series of conclusions. First, the extra chromosome 21 does not cause widespread strong dysregulation of gene expression. Second, genes on chromosome 21 are in fact disproportionately over-expressed in the +21 cells compared with genes on other chromosomes. So while regulatory feedback mechanisms are likely to exist, the data make it clear that fibroblasts and fetal heart cells do not completely compensate for this chromosomal aneuploidy. A similar conclusion has been reached by other groups studying the segmental trisomy 16 mouse ,33. Thirectively , and in MX1 encodes a large GTPase that forms organized multimeric structures in the cytoplasm. This protein is a well known interferon target gene, and studies both in cell culture and in vivo have documented that it is an essential component of the antiviral response. How this antiviral activity is exerted is not yet clear, and it appears that multiple steps in the viral life cycle are inhibited by p78MX1 [MX1 may also function outside of anti-viral defense. A link between MX1 and sporadic alopecia areata has been raised by a reasonably large genetic association study, including 165 alopecia areata cases and 510 controls, which showed that the +9959 intronic polymorphism conveyed a 1.79-fold increase in risk [MX1 is non-linearly over-expressed in +21 cells, and that this gene is activated in inflamed hair follicles in alopecia areata, add credence to MX1 as an alopecia areata susceptibility locus. Clinical studies using high-dose interferons to treat hepatitis and other conditions have reported alopecia as a common side effect, so intense and prolonged interferon signaling is clearly sufficient to produce alopecia [MX1 is playing an effector role in the arrest of the hair cycle in alopecia areata.y p78MX1 . But p78 in risk . Since aalopecia -40. AddiMX1 mRNA is modestly upregulated in normal fibroblasts during senescence is consistent with a report by Yoon et al., who used a cDNA microarray to screen for genes upregulated during replicative senescence, in a panel of 3 normal human fibroblast lines [MX1 mRNA and p78MX1 protein are much more strongly induced as +21 fibroblasts enter replicative senescence, it will be interesting to study the role of this gene, and of the interferon pathway more generally, in the process of premature aging and cell senescence. Various aspects of premature aging have been studied as phenotypes in DS [MX1, occurs with cellular immortalization in tissue culture [Our observation that st lines . Based oes in DS ,43, and es in DS . Howeveres in DS ,46. Rele culture , suggestDSCAM, collagen VI (COL6AI and COL6A2 genes), the calcineurin pathway modulator DSCR1, the SH3BGR gene, and others [SH3BGR was over-expressed on average in the +21 fetal hearts in our series, while DSCAM and the collagen VI genes were not . These cushions ,60,62 an to ours . It can turation . ExaminaDSCAM gene may be in a critical region for Tetralogy of Fallot [GART  is located substantially centromeric to DSCAM (41 Mb), outside this region.Gene expression measurements are only circumstantial evidence, and transgenic experiments in mice ,55, inclf Fallot ,54. GARTMX1 and other genes in the interferon pathway.From a more general viewpoint, our data and those from other laboratories cited above clearly show that human and mouse cells and tissues do not effectively compensate for simple chromosomal aneuploidies, and that this lack of compensation manifests as abnormal steady-state mRNA levels from different sets of genes in different cell types and tissues. For some genes the perturbations in mRNA expression resulting from these simple chromosome gains are non-linear, and it may be a general principle that this phenomenon can be explained by gene-gene interactions, as exemplified here by MX1 mRNA and p78MX1 protein in senescent fibroblasts with +21, and the over-expression of p78MX1 in inflamed hair follicles of alopecia areata, hyperactive interferon signaling is a candidate pathway for cell senescence and autoimmune disorders in DS. Additionally, the strong over-expression of the GART gene that we observe in fetal hearts with +21 suggests that abnormal purine metabolism should be investigated for a potential role in the cardiac defects associated with DS.Our results indicate over-expression of chromosome 21 genes in human cells and tissues with +21, and highlight different subsets of chromosome 21 genes as selectively over-expressed in different cell types. Our data further indicate that for some chromosome 21 genes this over-expression is non-linear (>1.5X). Based on the observed non-linear over-expression of DS – Down syndrome, +21 – trisomy 21.The author(s) declare that they have no competing interests.Chi-Ming Li carried out the experiments shown in all of the figures. Meirong Guo participated in carrying out the experiments in figures The pre-publication history for this paper can be accessed here:Genes over- or under-expressed in early passage fibroblasts with +21. ANOVA, p < .05.Click here for fileGenes over- or under-expressed in late passage fibroblasts with +21. ANOVA, p < .05.Click here for fileGenes over- or under-expressed in late passage compared to early passage fibroblasts, independently of +21 .Click here for fileGenes over- or under-expressed in fetal hearts with +21. ANOVA, p < .05.Click here for file


It has yet to be determined whether visual-tactile cross-modal plasticity due to visual deprivation, particularly in the primary visual cortex (V1), is solely due to visual deprivation or if it is a result of long-term tactile training. Here we conducted an fMRI study with normally-sighted participants who had undergone long-term training on the tactile shape discrimination of the two dimensional (2D) shapes on Mah-Jong tiles (Mah-Jong experts). Eight Mah-Jong experts and twelve healthy volunteers who were naïve to Mah-Jong performed a tactile shape matching task using Mah-Jong tiles with no visual input. Furthermore, seven out of eight experts performed a tactile shape matching task with unfamiliar 2D Braille characters.When participants performed tactile discrimination of Mah-Jong tiles, the left lateral occipital cortex (LO) and V1 were activated in the well-trained subjects. In the naïve subjects, the LO was activated but V1 was not activated. Both the LO and V1 of the well-trained subjects were activated during Braille tactile discrimination tasks.The activation of V1 in subjects trained in tactile discrimination may represent altered cross-modal responses as a result of long-term training. Visual-tactile cross-modal plasticity due to visual deprivation has been reported -5. For vThese findings indicate that cross-modal plasticity may be induced not only by sensory deafferentation but also by learning. Recently, audio-visual cross-modal plasticity due to learning has been shown while pianists observe piano playing . Well-trAnticipating a similar learning effect in visual-tactile cross-modal plasticity, we conducted fMRI studies in persons with normal sight who have had long-term tactile shape discrimination training on Mah-Jong tiles . Mah-JonP = 0.01, two-sample t-test). The mean accuracy during Braille discrimination by the seven subjects in the expert group was 72.9 ± 7.6%, which was significantly worse than the Mah-Jong discrimination (87.9 ± 7.0%) by the same subjects , and comparable with the accuracy scores of the control group during Mah-Jong discrimination .The mean percentage of correct responses during Mah-Jong tile discrimination was 88.8 ± 6.9% (mean ± SD) in the expert group, which was significantly better than the control group  and the left LO  of the expert group were more prominently activated  and the lateral occipital cortex (LO) on the left  and in V1  during the Braille task. The activation of V1 by the Braille task in the Expert group was significantly more prominent than that in the Control group by the Mah-Jong task  and twelve healthy volunteers who were naïve to Mah-Jong  participated in this study. Eleven subjects in the control group were right-handed and one was left-handed based on the Edinburgh handedness inventory . The lefIn each imaging session, a time-course series of 46 volumes was acquired using T2*-weighted, gradient echo, echo planar imaging (EPI) sequences with a 3.0 Tesla MR imager . The raw data were transferred to a parallel supercomputer  to reconstruct the consecutive 2D images using an algorithm of 2D fast Fourier transformation . Each volume consisted of 34 slices, each 3.5 mm thick with a 0.5-mm gap, to cover the entire cerebral and cerebellar cortex. The time interval between two successive acquisitions of the same image was 3000 ms, the echo time was 30 ms, and the flip angle was 90 degrees. The field of view (FOV) was 19 cm. The digital in-plane resolution was 64 × 64 pixels, with a pixel dimension of 2.97 × 2.97 mm. The magnetic shim was optimised such that a true in-plane resolution of 2.97 × 2.97 mm was realised. Tight but comfortable foam padding was placed around each subject's head to minimise head movement.For anatomical reference, T2-weighted fast spin echo images were obtained from each subject with location variables identical to those of the EPIs. In addition, high-resolution whole-brain MR images were obtained using a conventional T2-weighted, fast spin echo sequence. A total of 112 transaxial images were obtained. The in-plane matrix size was 256 × 256, slice thickness was 1.5 mm, and pixel size was 0.859 × 0.859 mm. These imaging data were utilized for anatomical normalization.The task design was described previously by Saito et al. . For tacAn fMRI session consisted of two rest and two task periods, each 30 s in duration, with the rest and task periods alternating. The subjects performed a tactile-tactile matching task with no visual input. The tactile matching task session was repeated twice. Prior to the fMRI session, the subjects were trained on the tactile discrimination tasks with the Mah-Jong tiles until their performance exceeded an accuracy level of 60%.The subjects lay in a supine position with both hands extended. Their left hand was placed on the button box, which was connected to a microcomputer for recording responses. The subjects closed their eyes throughout the session. During the 30 s rest periods, the experimenter touched the subject's foot every 6 s to signal to the subject that they should push the buttons alternately with the left index finger and the left middle finger. During the task periods, a block was manually placed on the subject's right palm every 6 seconds Figure . The bloTo determine if any training effect was material-specific, we conducted another functional MRI session with seven out of the eight Mah-Jong experts. The test materials were paired blocks of plastic Mah-Jong tiles, onto which two Braille characters had been pasted. The experimental conditions under which this session was carried out were identical to those used in the Mah-Jong session.The first 6 volumes of each fMRI session were discarded to allow for stabilisation of the magnetisation, and the remaining 40 volumes per session, a total of 80 volumes per subject, were used for analysis. The data were analysed using statistical parametric mapping  and implemented in Matlab  ,26. FollTwo levels of statistical analysis were conducted. First, we evaluated the individual task-related activation. Second, the summary data of each individual were incorporated into the second-level analysis using a random effects model ; this wat statistic (SPM{t}). The SPM{t} was transformed to the unit normal distribution (SPM{Z}). The threshold for SPM{Z} was set at Z > 3.09 and P < 0.05, with a correction for multiple comparisons at the cluster level for the entire brain [The signal was scaled proportionally by setting the whole-brain mean value to 100 arbitrary units. The signal time course for each subject was modelled using a box-car function convolved with a hemodynamic response function, session effect, and high-pass filtering 128 s). The explanatory variables were centred at 0. To test hypotheses about regionally-specific condition effects, the estimates for each model parameters were compared with the linear contrasts. First, we delineated the areas that were active during the tasks compared with those active during the rest periods of the same session. The resulting set of voxel values for each contrast constituted a statistical parametric map (SPM) of the  s. The et statistic (SPM{t}). The SPM{t} was transformed to the normal distribution unit (SPM{Z}). The threshold for SPM{Z} was set at Z > 3.09 and P < 0.05 with a correction for multiple comparisons at the cluster level for the occipital cortex [The weighted sum of the parameter estimates in the individual analysis constituted "contrast" images, which were used for the group analysis . The conl cortex .To determine whether any learning effect depicted by the between-group comparison was material-specific, Braille tactile discrimination by the expert group was evaluated at the local maximum foci detected by the between-group comparisons during Mah-Jong discrimination Table . The BraDNS carried out the MRI scanning, data analysis and drafted the manuscript.TO operated the MRI scanner.MH and YY participated in the task design.NS participated in the task design, data analysis, and revision of the manuscript.All authors read and approved the final manuscript.


Most of the Luci-eASCs remain in the iLNs and in the adipose tissue surrounding the inguinal LNs. A small proportion of Luci-eASCs can migrate to other locations within the lymphatic system and to other tissues and organs, having a preferential migration toward the intestine in colitic mice. Our results show that the infused Luci-eASCs protected 58% of the mice against induced colitis. Importantly, a correlation between the response to eASC treatment and a higher accumulation of eASCs in popliteal, parathymic, parathyroid, and mesenteric LNs were found. Altogether, these results suggest that IN administration of eASCs is feasible and may represent an effective strategy for cell therapy protocols with human adipose-derived MSCs in the clinic for the treatment of immune-mediated disorders.Mesenchymal stem cells (MSCs) have a large potential in cell therapy for treatment of inflammatory and autoimmune diseases, thanks to their immunomodulatory properties. The encouraging results in animal models have initiated the translation of MSC therapy to clinical trials. In cell therapy protocols with MSCs, administered intravenously, several studies have shown that a small proportion of infused MSCs can traffic to the draining lymph nodes (LNs). This is accompanied with an increase of different types of regulatory immune cells in the LNs, suggesting the importance of migration of MSCs to the LNs in order to contribute to immunomodulatory response. Intranodal (IN), also referred as intralymphatic, injection of cells, like dendritic cells, is being proposed in the clinic for the treatment of cancer and allergy, showing that this route of administration is clinically safe and efficient. In this study, we investigated, for the first time, the biodistribution and the efficacy of Luciferase Mesenchymal stem cells (MSCs) are multipotent adult stem cells capable of self-renewal and differentiation into multiple cell lineages of mesodermal origin such as chondrocytes, adipocytes, and osteocytes . These c+ eASCs (Luci-eASCs) after IN administration in steady state and following an acute inflammatory challenge in the colon with the haptenizing reagent trinitrobenzene sulfonic acid (TNBS). Strikingly, modulation of intestinal inflammation was observed. In parallel to this study, we also demonstrate that IN infusion of eASCs can modulate ongoing acute inflammatory responses in an experimental model of collagen-induced arthritis . All experiments were performed in accordance with the corresponding regulations regarding experimental animal welfare (RD 223/1998 and Directive 2010/63/EU protocols).Human samples were obtained after informed consent as approved by the Spanish Ethics Committee of reference for the site of tissue procurement . Human adipose tissue aspirates from healthy donors were processed as described elsewhere . All theAcc65I–BamHI sites. Internal ribosome entry site (IRES) element was isolated from pIRES2-EGFP  using SalI–MscI and cloned upstream H2B-CopGFP2i into SalI–Acc65I (Klenow blunted) sites. Luciferase was cut from pGL2 Basic , using XhoI–XbaI (Klenow blunted) restriction sites, and inserted upstream IRES-H2B-CopGFP2i into XhoI–SalI (Klenow blunted). CopGFP2i was then replaced by EGFP, from pEGFP-N1 (Clontech) using AgeI–NotI sites. The Luciferase-IRES-H2B-EGFP cassette was separated using BglII–NotI and cloned into BamHI–NotI sites of pRV retroviral vector to give the final pRV-Luciferase-IRES-H2B-EGFP bicistronic reporter construction. Transfection and generation of viral supernatants were performed using polyethylenimine (1 vol PEI:2 vol DNA). Viral supernatants were used for infection of eASCs. For infection, eASCs were seeded in 6-well plates and infected with retroviral particles concentrated when cell confluency was between 70 and 80%. Finally, the transduction was evaluated by flow cytometry , selecting clones with 90% expression of EGFP by cell sorting.Reporter Luciferase-EGFP bicistronic retroviral vector was constructed using standard cloning procedures. The H2B gene was amplified by PCR  and cloned into pCopGFP2i-N  using Luci-eASCs were defined according to the criteria of the International Society for Cellular Therapy . Luci-eABuffy coats were provided by the National Transfusion Centre of the Comunidad Autonoma of Madrid. Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation gradient using Ficoll-Paque Plus  following the supplier’s protocol. Purity was verified by flow cytometry.N-succinimidyl ester, Sigma-Aldrich, St. Louis, MO, USA) solution (107 PBMC per 200 µL of solution), and incubated under constant shaking at 37°C for 10 min. Reaction was stopped by adding ice-cold medium (RPMI + 10% FBS), and cells were washed three times with ice-cold phosphate buffer saline. Cells were then cultured overnight and one aliquot was used to set up and control the FL-1 voltage for CFSE. After resting overnight, CFSE-labeled PBMCs were activated with the Pan T Cell Activation Kit  following the manufacturer’s instructions. PBMCs (106 cells/well) were cultured in 24-well plates alone or with Luci-eASCs  in a total volume of 1 mL of RPMI + 10% FBS. The 1:25 ratio was chosen because it provided a high inhibitory effect, on the basis of previous studies (+/7-AAD− population (viable CD3 T lymphocytes) was determined by flow cytometry, according to loss of CFSE signal. Data were analyzed with the use of FCSExpress 4  and BD CellQuest™ Pro analysis (Becton Dickinson) softwares. CaliBRITE beads  were used before each assay to calibrate the cytometer.For CFSE labeling, PBMCs were washed extensively to remove fetal bovine serum (FBS), resuspended in a 10 µM CFSE . As controls, healthy mice infused with the Luci-eASC and mice treated with intrarectal 50% EtOH only (vehicle of the TNBS) and infused with Luci-eASC IN were used. Score of colitis  was monitored for 48 h.To induce colitis, male C57BL/6 mice (6–8 weeks old) were preimmunized on their shaved back with 1% of TNBS  in a mixture of acetone:olive (4:1 v/v). After 1 week, colitis was induced by intrarectal administration of 3 mg of TNBS in 50% ethanol (100 µL) per mouse. 1 h after TNBS administration, 3.2 × 10Bioluminescent imaging analysis was conducted at 48 h after the infusion of Luci-eASCs with the IVIS 200 imaging system .2 per steradian using Living Imaging 3.0 . For photon quantification, a region of interest was manually selected and kept constant within each experiment.Whole-body bioimaging analysis was done in anesthetized mice. The bioimaging analysis in main tissues and organs , secondary LNs  and the adipose tissue around the inguinal LNs, where the Luci-eASCs were injected, were determined immediately after culling the mice. Photons emitted were acquired as photons per s/cmBioluminescence signal was analyzed as percentage of light units per tissue relative to the total number of light units per mouse. Total bioluminescence signal for LNs, tissues, and organs were calculated as the sum of the light units in each tissue. Values of bioluminescence signal below 10,000 light units were considered negative.U test) were used (with Bonferroni adjustment). Analysis was performed using the software Stata 11  and GraphPad Prism 5.00 for Windows program .Data are presented as the interquartile range . Normal distribution was analyzed by the Shapiro-Wilks test. Non-parametric techniques , both in healthy [92.0 (70.3–99.2)%] and in TNBS-colitic mice [57.5 (46.8–79.6)%], whereas lower levels of the bioluminescence signals were detected in other organs and tissues analyzed  in both healthy [0.6 (0.3–2.6)%] and TNBS-colitic mice [2.8 (0.8–5)%] Figure . These rp = 0.0011] where the Luci-eASCs were injected. In contrast, very low levels of the bioluminescence signals [0.4 (0.2–1.3)%] were measured within the lymphatic system with no clear preference for any of the LNs analyzed  and in the tissues and organs . As shown in Figure s [0.4 0.–1.3%] weMoreover, high bioluminescence signals were also detected in the adipose tissue surrounding the iLNs . Increased bioluminescence signals, but not significant, were found in the liver [0.28 (0.04–0.80)%], spleen [0.10 (0.01–0.27)%], lungs [0.10 (0.02–0.30)%], and blood [0.14 (0.03–0.30)%] of the TNBS-colitic mice in comparison with the healthy mice  in comparison with the healthy mice . “Non-responder” mice were those TNBS-colitic mice that continued to lose weight 48 h after the infusion of the Luci-eASCs [−0.91 (−2.34 to −0.45)] in comparison with the colitic mice. According to this stratification criteria, when Luci-eASCs were administered IN, 15 out of the 26 mice did not lose weight (responder mice). This means that 58% of mice treated with Luci-eASCs had a positive response to the treatment with Luci-eASCs % for responders; 56.0 (40.0–72.3)% for non-responder mice] nor in the iLN-adipose tissue  in comparison with the heart [0.17 (0.03–0.30)%] of the responder mice but not in the non-responder mice [0.05 (0.03–0.57) vs. 0.090 (0.002–0.299)%, Figure Overall, these data indicate that, in responder mice, an increase amount of Luci-eASCs is located within the lymphatic system as suggested by the increase bioluminescence signals found in the popliteal, parathymic, parathyroid, and mesenteric LNs in comparison with the non-responder mice. However, in the tissues and organs analyzed these differences were not observed, although increased bioluminescence signals were found in the inflamed intestine.in vivo through lymphatic system and home to the LNs . This, obviously, might impact the bioavailability of the cells, their targeting to tissues and, probably, their long-term therapeutic efficacy % of the bioluminescence signal was found in the tissues and organs analyzed and, surprisingly, in TNBS-colitic mice, a significant increase of the total bioluminescence signal was found with in comparison with the healthy mice. This paralleled with a decrease in the bioluminescence signal in the iLNs and in the adipose tissue surrounding the iLNs of TNBS-colitic mice when compared to healthy mice Figures , suggestTrying to know if the presence of the MSCs at the inflammation site is necessary for the modulation of the immune responses, we investigated the correlation between the biodistribution of the Luci-eASCs and the modulation of the inflammation attending to the stratification criteria into “responders” and “non-responders” mice. These stratification criteria were done based on the body weights of the Luci-eASC-treated colitic mice. The increase amount of Luci-eASCs within the lymphatic system in the responder mice can be correlated with an improved immunomodulation Figure . This inIn summary, all these results suggest that the presence of the Luci-eASCs within the lymphatic system is responsible for their immunomodulatory effects. The IN administration of the Luci-eASCs allows the trafficking of a small proportion of cells to the inflamed intestine; although, a direct correlation with their biological effects could not be established. Further, these results confirm our hypothesis that the immunomodulatory effects of the eASCs may take place in the LNs where the immunoresponses are orchestrated.Based on these results and those reported in the accompanying manuscript , furtherThese data demonstrate the feasibility of using the IN route of administration to infuse eASCs for the treatment of intestinal inflammatory diseases since Luci-eASCs can modulate an acute intestinal inflammation.After IN administration, the majority of the Luci-eASCs remained in the inguinal LNs where the cells were infused and only lower levels of bioluminescence signals were found in tissues and organs related to the inflammation. After intestinal inflammation, an increased trafficking of the Luci-eASCs to the inflamed organs was found. In some instances, the increase amount of Luci-eASCs within the lymphatic system can be correlated with an improved immunomodulation. Taken together, these results indicate that the IN administration of eASCs represents a novel route for administering eASCs in cell therapy protocols.Institutional Animal Care and Use Committee at University of Albacete (Spain) and Spanish Ethics Committee of reference  for the site of tissue procurement.Conception and/or design of the work: ML-S, OD, JA, DB, JR, JB, WD, EL, and MG. Acquisition of data for the work: ML-S, PM-C, AE, RM, JA, EL, and MG. Analysis and interpretation of data for the work: ML-S, PM-C, AE, RM, OD, JA, DB, JR, JB, WD, EL, and MG. Manuscript writing: ML-S, PM-C, and MG. Revised manuscript for important intellectual content: AE, RM, OD, DB, JR, JB, and WD. All authors reviewed the manuscript and gave final approval for the work.PM-C, RM, OD, WD, and EL are full-time employees of TiGenix, DB is full-time employee of Grifols, and JA is full-time employee of Coretherapix.


The testosterone (T) and cortisol (C) hormones were measured at rest, immediately post-exercise and 15-min post-exercise. The Glucose (GLU), Free fatty acids (FFA) and enzyme lipase activity (ELP) were measured at rest, 15-min and 30-min exercise, immediately, 15-min and 30-min post-exercise. Significantly lower values were observed in FED compared to FAST with: C (nmol/L) from pre  to immediately post-exercise  and 15-min post-exercise  (p<0.05); and GLU at all times, with an exception at 15-min post-exercise. The testosterone/cortisol ratio (T/C) was significantly higher in the FED compared with FAST from pre  to 15-min post-exercise . No other significant differences were observed between conditions. We conclude that fasting prior to low intensity endurance exercise does not seem be advantageous, when it comes to fat loss, compared with the same exercise performed after a meal.The aim of the present study was to observe whether performing a low intensity endurance exercise following an overnight fasted (FAST) or fed (FED) condition promotes different cardiorespiratory, enzymatic and hormonal responses. Nine male physical active subjects, , randomly performed two sessions of 45 minutes’ low intensity exercise  interspersed by seven days, differentiated only in whether they were provided with a standardized meal or not. The oxygen consumption (VO The combination of diet and physical activity is recognized as a good strategy in preventing and fighting the increasing overweight and obesity rates in the world’s population –4. RecenHowever, performing aerobic exercise in fasting condition can induce fatigue and stimulation of the hypothalamic-pituitary-adrenal axis because of the low blood sugar levels ,10. ThisIt is necessary to understand the endogenous hormonal response to this strategy due to their influence in the recovery phase after exercise, by modulating anabolic and catabolic processes. Cortisol and Testosterone (T) are playing a significant role in metabolism of protein as well as carbohydrate metabolism ,11. In fFurthermore, testosterone is important as a stress biomarker. Due to the anabolic nature of T and the catabolic effects of C, it’s been suggested that the ratio between the two may reflect the body’s overall anabolic–catabolic balance. An increase in T, a decrease in C, or both, could indicate a potential status of anabolism, or in the other hand, a decrease in T, a increase in C, or both, could indicate a potential status of catabolism .To our knowledge only two studies were published on the effects of aerobic low-intensity exercise performed in the morning, following an overnight fast or fed condition ,14. HoweThe volunteers were nine Caucasian men, age 21.89 ± 2.52 years old, height 175.89 ± 5.16 cm, weight 72.10 ± 4.31 kg, estimated body fat 7.25 ± 2.11% that practiced the exercise in the treadmill at the first ventilator threshold velocity. The inclusion criteria in the present study were regular physical activity for at least 6 months. Participants which suffered from any metabolic, endocrine or osteo-articular conditions were excluded. The volunteers were recruited among the students of a master course class. Study procedures were agreed to by all participants who provided a written informed consent to participate in the study. The study complied with the Helsinki declaration and was approved by the Institutional Review Board of the University of Trás-os-Montes and Alto Douro, Vila Real, Portugal.2, COSMED®, Rome, Italy). In the ramp test the speed was 6 km.h-1 and inclination was increased 1% every minute until the participants were unable to walk without hand support. The lowest value of ventilation (VE) to oxygen consumption (VO2) ratio (VE/VO2) was used to mathematically calculate the velocity corresponding to the first ventilatory threshold.This was a cross-sectional study. All participants filled the Par-Q test and a medical history . After c2 device at a 30° head-of-bed elevation. Afterwards, the first blood sample was collected and the first condition of exercise was conducted (FAST or FED).The following two sessions were separated by a week and differed only in either fasting (FAST) or feeding (FED) conditions. The participants were led by a researcher to the laboratory and testing was started at 8.00 a.m. after 12 hrs of fasting. Resting metabolic rate (RMR) was measured through indirect calorimetric using the K4bIn the FAST exercise condition, participants performed 45 min of treadmill walking at a velocity corresponding to their individual first measured ventilatory threshold. In the FED exercise condition, participants consumed a standardized breakfast composed of a shake with 32g of carbohydrates (dextrose monohydrate), 24g of whey protein and 4g of fat (coconut oil), mixed with 35cl of water–subjects rested 30-min before starting the 45-min of walking. Breakfast intake was controlled by a technician.During the walking period, blood samples were collected at 15-min and 30-min and were also collected at 0-min, 15-min and 30-min post-exercise.2) on post-exercise recovery. During exercise and post-exercise, the VO2 was measured continuously breath-by-breath and then average in 20-seconds intervals. Breathing artifacts  were detected and automatically removed by the Cosmed software when values exceeded three standard deviations from the mean. All participants reported food intake of the previous day, before each exercise condition. Participants used a wearable device to measure cardiorespiratory responses (K4b2). This equipment was calibrated before each test according to the manufacturer´s indications . The ambient temperature and humidity were between 20–25°C and 40–65%, respectively, for all testing. The following were measured: VO2, carbon dioxide production, heart rate (HR), respiratory-exchange ratio (RER) at rest, during the 45-min walking and during 30-min after; testosterone (T) and cortisol during rest, immediately post and 15-min after exercise; glucose (GLU), free fatty acids (FFA) and the enzyme lipase (ELP) were measured at rest, 15-min and 30-min during walking, immediately post exercise, 15-min and 30-min afterwards. Substrate utilization (fat or carbohydrates) was calculated during the 45-min of walking using the formulas proposed by Frayn  condition does not seem to offer an advantage, as compared with performing the same exercise under a feeding [FED] condition. Moreover, based on Cortisol (C) levels, we also concluded that if a primary goal is to burn fat while, simultaneously, maintaining muscle mass, performing a low intensity aerobic exercise under a fasting condition might not be the best choice. These conclusions can only be extrapolated to a population with identical characteristics as that tested in the present study.S1 File(XLSX)Click here for additional data file.


We compared the protective effects of secure Chlorhexidine Gluconate (CHG)-containing dressings with those of non-antimicrobial transparent dressings.This prospective, comparative, single-center clinical study was conducted in a tertiary pediatric intensive care unit from October 2014 to March 2017. The inclusion criterion was catheterization of the jugular vein for ≥48 hour. The study was conducted in two phases. Non-antimicrobial standard dressings were applied both before and after the CHG- dressing phase to negate any coincidental temporal effect. During the standard-dressing phases, the dressings did not include any antimicrobial; transparent CHG-impregnated dressings were applied during the test phase. All patients were divided into two groups by the type of dressing applied (standard and CHG-containing dressings).p>0.05). The Catheter-Related Bloodstream Infection (CRBSI) rate was somewhat lower in the CHG-dressing group (20.6 vs. 26.5%), but the difference was not statistically significant (p>0.05). In the CHG-dressing group, CRBSIs caused by Gram-positive microorganisms totaled 0%, but the figure was 8.8% in the control group (p=0.028).The standard- and CHG-dressing groups contained 68 and 63 patients, respectively. The median durations of catheterization were 13 (8–22) and 14 (2–28) days, respectively (CHG dressings reduced CRBSIs caused by Gram-positive microorganisms. Central venous catheterization is common in Intensive Care Units (ICUs). Catheter-Related Bloodstream Infections (CRBSIs) are frequent complications of catheterization.2Chlorhexidine Gluconate (CHG) is frequently used to prevent CRBSIs; CHG in the soap used to bathe patients may reduce CRBSIs. CHG can also be used to sterilize the catheter site and wound dressings.3The type of dressing used may affect the infection rate.4We explored whether the regular use of a secure CHG-containing dressing  to protect the catheters of ICU patients reduced the frequency of CRBSIs.This was a prospective, comparative, single-center clinical study. It was performed between October 2014 and March 2017 in a tertiary care pediatric ICU. The study was conducted in two phases. Non-antimicrobial standard dressings were applied before and after the CHG-dressing phase to negate any coincidental temporal effect. Prior to this study, the non-antimicrobial “3M Tegaderm I.V. Dressing” was used for standard catheter care. We applied CHG dressings to half of all patients. Appropriate staff training was given prior to and during the changeover period. After the CHG-dressing phase, standard dressings were reintroduced. This study was approved by our local ethics committee (number: 2017/327). We found that a sample size of 61 patients per group would afford 80% power at the 5% significance level for the detection of a 20% difference between the two groups.The primary outcome was whether the new CHG dressing reduced the CRBSI rate.The secondary outcome was whether the CHG dressing affected the types of organisms causing CRBSIs.All patients with catheters in the jugular vein for ≥48 hour.Any existing BSI; known hypersensitivity to the study dressing or CHG per se; and/or eczema, rash, lesions, burns, or other skin conditions that might compromise skin integrity at the catheter insertion site; a hemodialysis catheter; combined jugular and femoral central venous catheters; and a catheter inserted prior to ICU admission.All catheterizations were performed by ICU pediatricians. Sedation and analgesia were achieved using ketamine (1 mg/kg) or fentanyl (1 µg/kg), plus midazolam (0.1 mg/kg). Before intervention, the access site was sterilized with a CH/alcohol solution . Non-tunneled, temporary central venous catheters  were used for all catheterizations. Double-lumen, 4-Fr, temporary central venous catheters were placed in patients of body weight <5 kg, and triple lumen 5- and 7-Fr catheters in patients weighing 5–20 kg and >20 kg, respectively.After fixation with sutures, the catheter site was coated with a transparent polyurethane film dressing  or a transparent dressing containing CHG and a hydrocolloid . The latter dressing is an adhesive, semipermeable, transparent polyurethane film incorporating a transparent gel pad containing 2% (w/w) CHG; the former dressing is an adhesive, semipermeable, transparent polyurethane film containing no antimicrobial. Dressings without CHG were replaced at 2-day intervals or as needed; CHG-containing dressings were replaced every seven days or as needed.5In patients catheterized for >2 days, a CRBSI was defined as one or more particular pathogens in one or more blood culture samples obtained between day three after catheterization and day one after removal of the catheter, along with a failure to identify any other source of infection, plus fever (>38°C), chills, and hypotension in a patient of any age or the presence of at least one of the following in patients aged <1 year: fever (>38°C), hypothermia <36°C), apnea, or bradycardia in addition to a failure to link positive culture findings to any other source of infection.°C, apneap-value <0.05 was considered statistically significant.All statistical analyses were performed with the aid of SPSS version 22.0 . Frequency data are expressed as counts (with percentages). Non-parametric continuous variables are expressed as medians (with the 25th–75th percentiles). The Mann-Whitney U-test was used to compare non-parametric variables between the two groups. Categorical variables were compared with the aid of the chi-squared test. In all tests, a The two groups did not differ significantly in terms of any of age, gender, underlying chronic disease, catheterization duration, number of catheter lumina, the side of catheterization, the vein used for catheterization, or the use (or not) of sonographic guidance during catheterization . The medThe CRBSI rates did not differ significantly between the groups (26.5% [n=18] in the control and 20.6 [n=13] in the test group). No CRBSIs caused by Gram-positive microorganisms were evident in the CHG-dressing group, but such infections caused 8.8% of all CRBSIs in the standard-dressing group (p=0.028). There was no significant difference between-group difference in the numbers of CRBSIs caused by either fungi or Gram-negative microorganisms .Acinetobacter baumannii was evident in the CHG-dressing group, but this bacterium caused 8.8% (n=5) of infections in the standard-dressing group  catheterization than after subclavian vein catheterization.Skin disinfection with CH and alcohol was recommended in 2011 by the Centers for Disease Control and Prevention (CDC) to prevent CRBSIs. We followed this recommendation for all patients. The same guidelines recommend the use of CHG- impregnated dressings.10CRBSI-related costs are reduced when CHG dressings are applied.Enterococcus (VRE) colonization and infection rates fell when CH soaps were used for bathing, but the Gram-negative infection rate was not affected.Staphylococcus aureus fell upon bathing with CH soap.A. baumannii infections. Hayashi et al.Acinetobacter strains. Our finding that no CRBSI caused by a Gram-positive microorganism was evident in the CHG-dressing group is significant.Afonso et al. reported that nosocomial infections caused by skin flora decreased when CH-containing soaps were used.Infection is also a common cause of catheter removal.The principal limitation of our study is the small sample size; we may have lacked adequate power to detect a significance between-group difference in the CRBSI rate. Another limitation is the lack of randomization.The CHG dressing reduced CRBSIs caused by the Gram-positive microorganism in pediatric patients.